Canine primary bronchiole epithelial cells and their application in preparation of immortalized cells

A technology of bronchioles and epithelial cells, applied in the biological field, can solve problems such as carcinogenicity, inappropriate pathogenic mechanism, and changing the original characteristics of primary cells

Active Publication Date: 2017-11-07
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

At present, the SV40 large T antigen is most widely used in the immortalization of cells. However, because the immortalized cells are carcinogenic and will change the original characteristics of the primary cells, the immortalized cells obtained by this method are used to study virus-infected cells. The pathogenic mechanism is inappropriate

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  • Canine primary bronchiole epithelial cells and their application in preparation of immortalized cells
  • Canine primary bronchiole epithelial cells and their application in preparation of immortalized cells
  • Canine primary bronchiole epithelial cells and their application in preparation of immortalized cells

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Embodiment Construction

[0043] Below in conjunction with specific embodiment. The experimental method that does not indicate specific condition in the embodiment, generally adopts routine condition, for example " Molecular Cloning Laboratory Handbook " (New York: Cold Spring Harbor Laboratory Press, 1989) (Sambrook et al.) described condition, or according to The method recommended by the manufacturer. As for the isolation and cell culture of canine primary bronchiole tissue, since there is no report on the isolation and culture of canine related tissue cells, we refer to the isolation method of human bronchial epithelial cells [Vos JB, van Sterkenburg MA, Rabe KF, et al.Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense. Physiol Genomics 2005,21(3):324-336].

[0044] (1) Isolation, primary cell culture and identification of canine bronchiole tissue

[0045] 1. Selection of Experimental Animals

[0046] The experiment...

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Abstract

The invention provides a canine primary bronchiole epithelial cell and its application in preparing immortalized cells, belonging to the field of biotechnology. The canine primary bronchiole epithelial cells were obtained from clean-grade male Beagle dogs aged 45 days. The canine tracheal tissue was separated, shredded and digested with collagenase and trypsin to obtain complete bronchiolar tissue, and the cells obtained after culture were further tested for the expression of keratin 18 by indirect immunofluorescence test, which confirmed the successful acquisition of canine primary bronchiolar epithelial cells. A eukaryotic expression vector pEGFP‑hTERT containing telomerase gene (hTERT) was constructed. After transfection into canine primary bronchiole epithelial cells, the hTERT gene was highly expressed, and the bronchiole epithelial cells could be stably subcultured for at least 20 generations. The canine immortalized bronchiole epithelial cells prepared by the invention provide important biological materials for studying the infection mechanism of canine pathogens such as canine influenza virus.

Description

technical field [0001] The invention relates to canine primary bronchiole epithelial cells and their application in preparing immortalized cells, belonging to the field of biotechnology. Background technique [0002] With the improvement of human civilization, the disease research of companion animals such as dogs has long been a focus of veterinary research. For dogs, infectious diseases mainly occur in the digestive tract and respiratory tract, and respiratory diseases are common throughout the year, and they are the most harmful to dogs. At present, microorganisms such as a variety of viruses and bacteria are considered to be the pathogens that cause canine respiratory diseases, such as the canine influenza virus that broke out in 2004 and was subsequently reported. However, compared with livestock and poultry pathogens, research on canine pathogens is relatively weak, especially what role these pathogens play in the occurrence of canine respiratory diseases and how the ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N15/70C12N15/66
Inventor 刘永杰谢星庞茂达郁磊林焱赵艳兵梁姗马可
Owner NANJING AGRICULTURAL UNIVERSITY
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